Abstract
Flavin-based electron transfer bifurcation is emerging as a fundamental and powerful mechanism for conservation and deployment of electrochemical energy in enzymatic systems. In this process, a pair of electrons is acquired at intermediate reduction potential (i.e. intermediate reducing power), and each electron is passed to a different acceptor, one with lower and the other with higher reducing power, leading to "bifurcation." It is believed that a strongly reducing semiquinone species is essential for this process, and it is expected that this species should be kinetically short-lived. We now demonstrate that the presence of a short-lived anionic flavin semiquinone (ASQ) is not sufficient to infer the existence of bifurcating activity, although such a species may be necessary for the process. We have used transient absorption spectroscopy to compare the rates and mechanisms of decay of ASQ generated photochemically in bifurcating NADH-dependent ferredoxin-NADP+ oxidoreductase and the non-bifurcating flavoproteins nitroreductase, NADH oxidase, and flavodoxin. We found that different mechanisms dominate ASQ decay in the different protein environments, producing lifetimes ranging over 2 orders of magnitude. Capacity for electron transfer among redox cofactors versus charge recombination with nearby donors can explain the range of ASQ lifetimes that we observe. Our results support a model wherein efficient electron propagation can explain the short lifetime of the ASQ of bifurcating NADH-dependent ferredoxin-NADP+ oxidoreductase I and can be an indication of capacity for electron bifurcation.
Highlights
Flavin-based electron transfer bifurcation is emerging as a fundamental and powerful mechanism for conservation and deployment of electrochemical energy in enzymatic systems
We extended the transient absorption spectroscopy (TAS) approach that was successful in NADH-dependent ferredoxin-NADPϩ oxidoreductase I from Pyrococcus furiosus (NfnI) to detect anionic flavin semiquinone (ASQ) in each of our systems and discovered ASQ lifetimes ranging over 2 orders of magnitude, demonstrating that short lifetime alone does not necessarily imply thermodynamic instability of the flavin ASQ per se
We attribute the decay of ASQ to reoxidation in all four cases, we propose that it represents charge recombination (CR) in Nitroreductase from Enterobacter cloacae (NR), NADH oxidase from Thermus thermophilus (NADOX), and Fld, as opposed to the electron transfer (ET) into a chain of electron carrier cofactors that can propagate the electron further, which has been demonstrated in the case of NfnI [9]
Summary
TAS as a function of time after a short pulse of irradiation at 400 nm was used to probe for possible formation of ASQ (Fig. 1). Flavin fluorescence is strongly quenched in Fld, and this is consistent with onset of absorbance at 370 nm within the time resolution of our instrument (supplemental Fig. S2d), presumably due to electron abstraction from the nearby Trp to form ASQ [25]. The addition of 2-(phenylamino)benzoic acid (PAB) or 2aminobenzoic acid (AB) results in rapid decay of ASQ and restoration of OX that occurs on a time scale comparable with formation of ASQ and greatly diminishes the extent to which ASQ accumulates (Fig. 4) In both cases, a small fraction of the sites appear to retain a comparatively long-lived ASQ despite the large concentration of donors used compared with their Kd values.
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